Polyphenolic additives in sequencing by synthesis
AssigneeIsoPlexis Corporation
InventorsJerzy Olejnik, Michel Georges Perbost
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Abstract
The invention relates to methods, compositions, devices, systems and kits as described including, without limitation, reagents and mixtures for determining the identity of nucleic acids in nucleotide sequences using, for example, sequencing by synthesis methods. In particular, the present invention contemplates the use of polyphenolic compounds, known as antioxidant additives, to improve the efficiency of Sequencing-By-Synthesis reactions. For example, gallic acid (GA) is shown herein to be one of many exemplary SBS polyphenolic additives.
Claims18
A method of incorporating labeled nucleotides, comprising:
- a) providing
- i) a plurality of nucleic acid primers and template molecules,
- ii) a polymerase,
- iii) a cleave reagent comprising a reducing agent and the polyphenolic compound gentisic acid, and
- iv) a plurality of nucleotide analogues wherein at least a portion of said nucleotide analogues is labeled with a label attached through a cleavable disulfide linker to the base;
- b) hybridizing at least a portion of said primers to at least a portion of said template molecules so as to create hybridized primers;
- c) incorporating a first labeled nucleotide analogue with said polymerase into at least a portion of said hybridized primers so as to create extended primers comprising an incorporated labeled nucleotide analogue;
- d) detecting said incorporated labeled nucleotide analogue;
- and
- e) cleaving the cleavable linker of said incorporated nucleotide analogues with said cleave reagent.
A method of incorporating labeled nucleotides, comprising:
- a) providing
- i) a plurality of nucleic acid primers and template molecules,
- ii) a polymerase,
- iii) a cleave reagent comprising a reducing agent and the polyphenolic compound pyrocatechol, and
- iv) a plurality of nucleotide analogues wherein at least a portion of said nucleotide analogues is labeled with a label attached through a cleavable disulfide linker to the base;
- b) hybridizing at least a portion of said primers to at least a portion of said template molecules so as to create hybridized primers;
- c) incorporating a first labeled nucleotide analogue with said polymerase into at least a portion of said hybridized primers so as to create extended primers comprising an incorporated labeled nucleotide analogue;
- d) detecting said incorporated labeled nucleotide analogue;
- and
- e) cleaving the cleavable linker of said incorporated nucleotide analogues with said cleave reagent.
A method of incorporating labeled nucleotides, comprising:
- a) providing
- i) a plurality of nucleic acid primers and template molecules,
- ii) a polymerase,
- iii) a cleave reagent comprising a reducing agent and the polyphenolic compound pyrogallol, and
- iv) a plurality of nucleotide analogues wherein at least a portion of said nucleotide analogues is labeled with a label attached through a cleavable disulfide linker to the base;
- b) hybridizing at least a portion of said primers to at least a portion of said template molecules so as to create hybridized primers;
- c) incorporating a first labeled nucleotide analogue with said polymerase into at least a portion of said hybridized primers so as to create extended primers comprising an incorporated labeled nucleotide analogue;
- d) detecting said incorporated labeled nucleotide analogue;
- and
- e) cleaving the cleavable linker of said incorporated nucleotide analogues with said cleave reagent.
Dependencies are inferred from the claim text.